In contrast to previous reports, removal of the hydrophobic 22-29 group of amino acid residues from glucagon does not result in loss of biological actions of the hormone on adenylate cyclase in hepatic membranes. Glucagon1-21 is a full agonist with a potency 1/1000 that of the native hormone. The implication of these findings is that the hydrophobic C-terminal region of glucagon is necessary for tight binding of the hormone to its receptors but not for action. The alpha amino and epsilon amino groups of glucagon have been amindated with a series of alkyl imidates. Alkyl amidation of the epsilon amino group yields glucagon derivatives that are as potent as the native hormone whereas amindation of the alpha amino N-terminal residue results in partial agonists. Selective incorporation of reporter imido groups in the epsilon amino group has given new derivatives of glucagon that can now be used for affinity labeling of the glucagon receptor.